The purpose of this thesis was to investigate the role of lactate in the regulatory volume response of mammalian skeletal muscle to hypertonic challenge-induced cell shrinkage. It was demonstrated that adult mice skeletal muscle single fibres responded to increased extracellular osmolarity in a dose-dependent manner when exposed to NaCl or sucrose challenge. This regulatory response to sucrose and NaCl however was abolished when cells were pre-treated with bumetanide, a specific sodium-potassium-chloride cotransport (NKCC) inhibitor, demonstrating that the NKCC is primarily responsible for eliciting a regulatory volume increase (RVI). When cells were exposed to NaLac treatment, bumetanide incubation did not significantly diminish the ability of the cells to recover volume. Furthermore, these cells lost less volume compared to NaCl or sucrose control. Inhibiting the single muscle fibres with either monocarboxylate transport (MCT) inhibitor phloretin or pCMBS resulted in significantly greater volume loss and impaired volume recovery. Combined MCT inhibition of phloretin or pCMBS with NKCC inhibition (bumetanide) led to unexpected findings, whereby the cells lost very little volume. These data suggest that while skeletal muscle fibres may utilize the NKCC to regulate volume, the ability for these cells to employ the most efficient means of volume regulation involves the inclusion of lactate as well via MCT uptake. / NSERC

No description available.

The ability of oocytes and early cleavage-stage embryos to regulate their volume is essential to avoid developmental arrests at in vivo-osmolarities. This is accomplished primarily via GLYT1-mediated glycine transport into the cells. GLYT1 activity has previously been shown to be absent in freshly isolated oocytes but becomes activated ~3-4 hours after oocyte maturation has been initiated either by isolation from ovarian follicles in vitro or following an ovulatory stimulus in vivo. GLYT1 activity then persists until the 4-cell stage of preimplantation embryo development. GLYT1 has been shown to spontaneously activate in oocytes that are isolated from follicles either as denuded oocytes or as cumulus-oocyte complexes (COCs), this implies that GLYT1 activity is suppressed in intact follicles in the ovary. However, it is not known how GLYT1 activity is suppressed within the ovarian follicle or how initial GLYT1 activation occurs. The activation of independent cell volume regulation in oocytes first involves the release of the strong adhesion between the oocyte and zona pellucida (ZP) followed by secondary GLYT1 activation. These two processes have been shown to occur spontaneously in fully grown oocytes following isolation from ovarian follicles, however, it is not known whether small growing oocytes within ovarian follicles already possess the ability to detach from the ZP and activate GLYT1. An osmotic assay was used to determine when during oogenesis oocytes are first able to detach from the ZP while the ability to activate GLYT1 was determined by measuring [3H]-glycine uptake into oocytes. I found that oocytes acquire the ability to detach from the ZP when they are nearly fully grown and similarly, that high levels of GLYT1 activity first develop in isolated oocytes during the late stages of oogenesis. Furthermore, I showed that SLC6A9 protein (GLYT1 transporter protein) and Slc6a9a transcripts steadily increased during oogenesis with SLC6A9 protein becoming localized to the oocyte plasma membrane during oocyte growth with predominant membrane localization apparent in fully grown oocytes. Taken together, these results suggest that oocytes become able to detach from the ZP and fully activate GLYT1 towards the end of oogenesis but that these processes remain suppressed in the ovarian follicle. Intact and punctured antral follicles were used as a model to examine the potential mechanism(s) mediating GLYT1 suppression before ovulation is triggered. Using these models, I found that GLYT1 activity remains suppressed within preovulatory antral follicles in contrast to the spontaneous GLYT1 activation that occurred in isolated denuded oocytes or within COCs. Recently, the mechanism mediating oocyte maintenance of prophase I arrest within the ovarian follicle was elucidated and was shown to depend on the release of Natriuretic Peptide Precursor C (NPPC) from mural granulosa cells (MGCs) into follicular fluid which binds to NPR2 guanylate cyclases on cumulus cells stimulating the production of cyclic GMP (cGMP) within these cells. Diffusion of cGMP from cumulus granulosa cells to the oocyte via gap junctions is required to maintain meiotic arrest. Although GLYT1 activation and meiotic resumption are both suppressed in antral follicles prior to the ovulatory trigger and these two processes occur simultaneously following oocyte isolation from ovaries, I have shown here that GLYT1 suppression within the preovulatory antral follicle is mediated by a mechanism distinct from the gap junction-dependent NPPC-cGMP pathway controlling meiotic arrest. I also showed for the first time a direct requirement for meiotic arrest of both gap junctions between granulosa cells (composed of connexin-43) and between the inner layer of cumulus granulosa cells and the oocyte (composed of connexin-37). Since I showed that GLYT1 was suppressed in isolated antral follicles but not COCs, I hypothesized that MGCs are required to maintain low GLYT1 activity in antral follicles. I showed here that MGCs isolated from preovulatory antral follicles were sufficient to maintain GLYT1 suppression in co-cultured COCs, but not denuded oocytes. Furthermore, I found that GLYT1 activity was suppressed in COCs cultured in conditioned medium from MGC cultures. Thus, GLYT1 activity appears to be suppressed within the ovary prior to the ovulatory LH-stimulus likely by an unidentified inhibitory signal within the ovarian follicle originating from the MGCs and propagated by a gap junction-independent mechanism involving multiple cell types in the follicle.

Reproductive science plays an important role in conservation biology. Quantitative studies of basic reproductive biology in wildlife are critical for the development of successful assisted reproductive technologies. Investigation of the volume regulatory mechanism of spermatozoa could produce options to improve the cryopreservation of spermatozoa and provide a non-hormonal contraceptive option for men, both of which could have significant impacts on global biodiversity preservation. Volume regulation of somatic cells involves the movement of osmolytes through various channels, including potassium channels. The potassium channels involved in volume regulation of human, monkey, and murine spermatozoa were investigated. Flow cytometry was used to gauge the sensitivity of the volume regulatory process of spermatozoa to various potassium channel inhibitors and a simultaneous hypotonic challenge. Channels potentially involved in regulatory volume decrease of spermatozoa varied with species but included voltage-gated (Kv) channels 1.4, 1.5, 1.7, 4.1, 4.2 and 4.3 as well as TWIK1, TWIK2, TASK1, TASK2, TASK3, TREK2 , and minK. The presence of some of these channels was confirmed by western blotting and immunocytochemistry. Changes in the motility patterns of human and monkey spermatozoa in the presence of potassium channel inhibitors during hypotonic stress were also observed, suggesting a relationship between volume regulation and motility. To evaluate potential organic osmolytes involved in, and compare effects of CPAs on, volume regulation, the isotonicity of murine epididymal spermatozoa was measured using a null point method. Spermatozoa were then exposed to high concentrations of various osmolytes and cryoprotective agents in isotonic medium to evaluate which compounds were able to penetrate the sperm plasma membrane. The osmotic responses of spermatozoa from strains of mice known to have spermatozoa of high (B6D2F1) and low (C57BL6) post-thaw fertility were compared during various osmotic challenges in various media. These experiments indicated that spermatozoa from B6D2F1 mice may have better volume regulation capabilities than spermatozoa from C57BL6 mice, suggesting that better post-thaw fertility of murine spermatozoa could be influenced by the volume regulatory process. The knowledge gained from these experiments could contribute to improved sperm handling and preservation techniques and be used to develop non-hormonal male contraceptives based on inhibiting volume regulation.

Sarcolemmal chloride channels and associated cell volume regulatory pathways have been shown to be important in local ischemic preconditioning (IPC) induced protection against myocardial ischemia/reperfusion injury. Similarities between intracellular pathways in remote (rIPC) and classic IPC suggest that these mechanisms may also play an important role in rIPC. rIPC protected cultured rabbit ventricular cardiomyocytes against necrosis caused by 75 minutes simulated ischemia followed by 60 minutes simulated reperfusion. The protective effect was abolished by chloride channel blockade using 50 μM indanyloxyacetic acid 94 (IAA-94). rIPC also reduced peak cardiomyocyte swelling during exposure to 200 mOsm hypo-osmotic buffer. The reduction in peak swelling was also abolished by IAA-94. These results suggest that the protective effect of rIPC is achieved, at least in part, by enhancing cell volume regulation and that this effect is dependent on the availability of chloride channels in a similar fashion to local IPC.

Sarcolemmal chloride channels and associated cell volume regulatory pathways have been shown to be important in local ischemic preconditioning (IPC) induced protection against myocardial ischemia/reperfusion injury. Similarities between intracellular pathways in remote (rIPC) and classic IPC suggest that these mechanisms may also play an important role in rIPC. rIPC protected cultured rabbit ventricular cardiomyocytes against necrosis caused by 75 minutes simulated ischemia followed by 60 minutes simulated reperfusion. The protective effect was abolished by chloride channel blockade using 50 μM indanyloxyacetic acid 94 (IAA-94). rIPC also reduced peak cardiomyocyte swelling during exposure to 200 mOsm hypo-osmotic buffer. The reduction in peak swelling was also abolished by IAA-94. These results suggest that the protective effect of rIPC is achieved, at least in part, by enhancing cell volume regulation and that this effect is dependent on the availability of chloride channels in a similar fashion to local IPC.

The choroid plexuses are largely responsible for cerebrospinal fluid (CSF) secretion and therefore play a fundamental role in brain homeostasis. The membrane proteins involved in CSF secretion are not fully known. Several electroneutral transporters have been identified by molecular methods in choroid plexus epithelial cells but there is a lack of functional data to support their expression making it impossible to elucidate their role in CSF secretion fully. The activity of many of these transporters can be observed in cell volume regulation. Thus, the main aim of the present study was to determine the ability of mammalian choroid plexus epithelial cells to regulate their volume in response to anisosmotic challenge and to investigate the transporters involved.Experiments were performed on cells isolated from the mouse fourth ventricle choroid plexus. Cells were isolated using a combination of manual perturbation, the enzyme dispase and a Ca2+ free incubation to disrupt tight junctions. Cell volume was measured using a video-imaging method. Cells used in this study were all of a similar morphology and had a mean volume of 0.71 pL.Cells exhibited a HCO3- dependent regulatory volume increase (RVI) in response to hypertonic challenge. Strong evidence is presented that the Na+/H+ exchanger (NHE1) and the Cl-/HCO3- exchanger (AE2) contribute to the RVI but the Na+K+2Cl- cotransporter (NKCC1) and the epithelial Na+ channel (ENaC) do not. Choroid plexus cells exhibit a HCO3- dependent regulatory volume decrease (RVD) in response to hypotonic challenge. The RVD was unaffected by DIOA (an inhibitor of KCC activity), the K+ channel inhibitors TEA+, Ba2+ or 4AP or the Cl- channel inhibitors DIDS or NPPB. However removal of extracellular Ca2+ completely abolished cell swelling in response to hypotonic challenge. This sensitivity of volume change to Ca2+ was specific to cell swelling as cell shrinkage in hypertonic artificial CSF was unaffected by removal of extracellular Ca2+.Thus functional evidence is presented to further elucidate the role of several proteins in the choroid plexus cell volume regulatory response to anisosmotic challenge.

Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society. A volume-regulated anion channel (VRAC) has been electrophysiologically characterized in innumerable mammalian cell types. VRAC is activated by cell swelling and mediates the volume regulatory efflux of Cl− and small organic solutes from cells. Two groups recently identified the mammalian leucine-rich repeat containing protein LRRC8A as an essential VRAC component. LRRC8A must be coexpressed with at least one of the other four members of this gene family, LRRC8B-E, to reconstitute VRAC activity in LRRC8−/− cells. LRRC8 genes likely arose with the origin of chordates. We identified LRRC8A and LRRC8C-E orthologs in the zebrafish genome and demonstrate that zebrafish embryo cells and differentiated adult cell types express a swelling-activated Cl− current indistinguishable from mammalian VRAC currents. Embryo cell VRAC currents are virtually eliminated by morpholino knockdown of the zebrafish LRRC8A ortholog lrrc8aa. VRAC activity is fully reconstituted in LRRC8−/− human cells by coexpression of zebrafish lrrc8aa and human LRRC8C cDNAs. lrrc8aa expression varies during zebrafish embryogenesis and lrrc8aa knockdown causes pericardial edema and defects in trunk elongation and somatogenesis. Our studies provide confirmation of the importance of LRRC8A in VRAC activity and establish the zebrafish as a model system for characterizing the molecular regulation and physiological roles of VRAC and LRRC8 proteins.

A loss of sarcolemmal dystrophin was observed by immuno-fluorescence studies in rabbit hearts subjected to in situ myocardial ischemia and by immuno-blotting of the Triton soluble membrane fraction of isolated rabbit cardiomyocytes subjected to in vitro ischemia. This ischemic loss of dystrophin was a specific event in that no ischemic loss of sarcolemmal α-sarcoglycan, γ-sarcoglycan, αDG, or βDG was observed. The maintenance of sarcolemmal βDG (43 Kd) during ischemia was interesting in that dystrophin binds to the C-terminus of βDG. However, during late in vitro ischemia, a 30 Kd band was observed that was immuno-reactive for βDG. Additionally, this 30 Kd-βDG band was observed in rabbit myocardium subjected to autolysis. Finally, the 30 Kd-βDG was observed in the purified sarcolemmal fraction of rabbit cardiomyocytes subjected to a prolonged period of in vitro ischemia, confirming the sarcolemmal localization of this band. The potential patho-physiologic significance of this band was indicated by the appearance of this band at 120-180 min of in vitro ischemia, directly correlating with the onset of irreversible injury, as manifested by osmotic fragility. Additionally the appearance of this band was significantly reduced by the endogenous cardioprotective mechanism, in vitro ischemic preconditioning, which delays the onset of osmotic fragility. In addition to dystrophin, βDG binds caveolin-3 and Grb-2 at its C-terminus. The presence of Grb-2 and caveolin-3 in the membrane fractions of oxygenated and ischemic cardiomyocytes was determined by Western blotting. An increase in the level of membrane Grb-2 and caveolin-3 was observed following ischemic preconditioning as compared to control cells. The formation of this 30 Kd-βDG degradation product is potentially related to the transition from the reversible to the irreversible phase of myocardial ischemic cell injury and a decrease in 30 Kd-βDG might mediate the cardioprotection provided by ischemic preconditioning.

No description available.